Novel 1-hydroxy phenothiazinium-based derivative protects against bacterial sepsis by inhibiting AAK1-mediated LPS internalization and caspase-11 signaling

Sepsis is a life-threatening syndrome with disturbed host responses to severe infections, accounting for the majority of death in hospitalized patients. However, effective medicines are currently scant in clinics due to the poor understanding of the exact underlying mechanism. We previously found that blocking caspase-11 pathway (human orthologs caspase-4/5) is effective to rescue coagulation-induced organ dysfunction and lethality in sepsis models. Herein, we screened our existing chemical pools established in our lab using bacterial outer membrane vesicle (OMV)-challenged macrophages, and found 7-(diethylamino)-1-hydroxy-phenothiazin-3-ylidene-diethylazanium chloride (PHZ-OH), a novel phenothiazinium-based derivative, was capable of robustly dampening caspase-11-dependent pyroptosis. The in-vitro study both in physics and physiology showed that PHZ-OH targeted AP2-associated protein kinase 1 (AAK1) and thus prevented AAK1-mediated LPS internalization for caspase-11 activation. By using a series of gene-modified mice, our in-vivo study further demonstrated that administration of PHZ-OH significantly protected mice against sepsis-associated coagulation, multiple organ dysfunction, and death. Besides, PHZ-OH showed additional protection on Nlrp3−/− and Casp1−/− mice but not on Casp11−/−, Casp1/11−/−, Msr1−/−, and AAK1 inhibitor-treated mice. These results suggest the critical role of AAK1 on caspase-11 signaling and may provide a new avenue that targeting AAK1-mediated LPS internalization would be a promising therapeutic strategy for sepsis. In particular, PHZ-OH may serve as a favorable molecule and an attractive scaffold in future medicine development for efficient treatment of bacterial sepsis.


General information
Unless otherwise noted, all reagents were purchased from commercial vendors and used without further treatments. All moisture-sensitive reactions were carried out in dried solvents under a nitrogen atmosphere. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz (Bruker Avance III NMR Spectrometer) for 1 H NMR and 100 MHz for 13 C NMR. Mass spectra (MS) were performed on a Waters ACQUITY UPLC M-Class spectrometer and High Resolution Mass Spectra (HRMS) were acquired on a Thermo Finnigan MAT 95 KL mass spectrometer. High-performance liquid chromatography (HPLC) chromatograms were acquired a Waters ACQUITY Arc system with a GL Sciences Inertsil ODS-4 column (150 mm × 4.6 mm, 5 μm) and an auto-sampler.

Synthesis and Characterization
Scheme S1. Schematic Illustration for the preparation of compound 5 (PHZ-OH)

3-(ethoxymethoxy)-N,N-diethylaniline (2)
To a stirred solution of 3-(diethylamino)phenol (compound 1, 1.012 g, 6 mmol) in 20 mL dried THF was added NaH (0.36 g, 9 mmol) at 0 o C, the reaction mixture was stirred in an ice-box for 30 min. Then (chloromethoxy)ethane (0.7 mL, 7.2 mmol) was added slowly with stirring. The reaction mixture was warmed up to room temperature and stirred for another 6 h, then a solution of saturated NH4Cl (3 mL) was added to quench the reaction. After removal of the solvent, the mixture was re-dissolved in 20 mL ethyl acetate, washed by saturated brine, and then the organic layer was dried and concentrated to leave oil. The residue was purified by flash chromatography with diethyl ether/hexane as eluent to afford compound 2 as a colorless oil (0.91 g, 68%

2-amino-5-(diethylamino)benzenesulfonoperoxothioic O-acid (3) (ref)
Compound 3 was prepared as reported. Briefly, To a solution of N,N-diethyl-p-phenylenediamine (1 eq) in water and methanol (H2O/MeOH 4:1) was added ZnCl2 (6.3 M in water, 1.0 eq) and HCl (10 mol/L, 1 eq). Then the mixture was cooled to 0 o C with an ice-water box, and an aqueous solution of newly prepared K2Cr2O7 (0.5 mol/L, 3.0 eq) and Na2S2O3 (3 mol/L, 2.0 eq) were added, and the mixture was stirred for another 3 h. After warmed up to room temperature (RT), the reaction mixture was stirred for a further 1 h, and then the mixture was filtered, washed with water and acetone, and

S3
dried under vacuum to afford compound 3 as gray solid, which was used as such in the next step.

3,7-bis(diethylamino)-1-hydroxyphenothiazin-5-ium chloride (5)
Compounds 2 (55.8 mg, 0.25 mmol) and 3 (0.14 g, 0.5 mmol) were dissolved in methanol and ethyl acetate (6 mL, MeOH/Ethyl Acetate 4:1), and the mixture was stirred at 80 o C for a while. Silver carbonate (0.14 g, 0.5 mmol) was then added slowly under reflux to generate a deep blue solution. The reaction mixture was refluxed with stirring for 2 h, and then the mixture was allowed to cool to RT and filtered by celite, and next the solid was washed by methanol and DCM. The filtrate was concentrated to leave a residue, and purified by flash chromatography to afford a deep blue solid, which was used as such in the next step.
To a stirred solution of the above obtained compound 4 (40.0 mg, 0.094 mmol) in a dried solution of DCM and THF (6 mL, 1:1) was added HCl (0.1 mL, 5N in MeOH), and the mixture was refluxed for 2 h. After cooled to room temperature, the solvent was removed under reduced pressure, and the residue was purified by flash chromatography to afford compound 5 as a blue solid (